Bioorthogonal labeling involves the metabolic or genetic incorporation of a biomolecule of interest containing a functional group (also called chemical reporter) into live cells and organisms, and subsequent labeling with a bioorthogonal probe that carries the specific reaction partner, i.e., that has certain functional group for reaction with the abiotic functional group of the biomolecule. Recently, there has been a rapidly emerging interest in exploiting bioorthogonal probes, in particular those which can display emission turn-on after the labeling, i.e., which remain essentially non-emissive as long as the appended bioorthogonal group is intact but show emission turn-on upon the specific labeling. Their use could eliminate the need for stringent washouts to remove unreacted probes, improve the signal-to-noise ratio, and offer the opportunity to monitor biological processes in real time.
Although there are a number of fluorescent bioorthogonal probes available in the market, all of them are organic dyes. They show strong fluorescence but their applications are limited by several key factors such as high photobleaching rates, substantial self-quenching, high pH dependence, and short-lived fluorescence that is not compatible with fluorescence-lifetime imaging microscopy (FLIM). There are a small number of organic bioorthogonal probes that show fluorogenic labeling properties, i.e., fluorescence will be turned on after the bioorthogonal labeling. However, the design of these probes is based on the use of an azide unit, which quenches the fluorescence of the compounds before their reactions with alkynes. The largest limitation of these fluorogenic probes is that the azide unit is susceptible to reduction by thiols, which commonly exist in living systems (e.g. the intracellular concentration of glutathione in human cells usually ranges from 0.5 to 10 mM). This limits the applications of these azide-containing fluorogenic probes because the emission could be turned on in the absence of the genuine chemical reporter, i.e., in the absence of the abiotic functional group.
Since the concept of bioorthogonal chemistry appeared in the literature less than twenty years ago, a number of chemical reporters and fluorescent bioorthogonal probes have been designed and produced. However, most of the probes developed so far are organic dyes that are already highly fluorescent before the specific labeling, and hence stringent washouts are required after the staining. The use of probes that show emission turn-on after the bioorthogonal labeling provided so far is limited for several reasons as mentioned above. Thus, there remains a strong need for bioorthogonal probes that show emission turn-on only after the specific labeling, which are further photostable, do not suffer from self-quenching, and allow for a sufficient enhancement of emission after the labeling reaction which emission has a long lifetime, e.g. which is compatible with FLIM and, for example, suitable for detection of cell structures and measurement of metabolic activities in the native biological environments, for disease diagnosis, and for identification of therapeutics.